RU2242471C1 - Derivatives of 2-h-1-benzopyrane-2-one eliciting anti-calcium activity - Google Patents

Abstract

FIELD: organic chemistry, heterocyclic compounds, medicine, cardiology.

SUBSTANCE: invention relates to new chemical compounds of heterocyclic row eliciting the expressed anti-calcium activity and representing derivatives of 2-H-1-benzopyrane-2-one of the general formula (I):

wherein R and R₁ have values given in the invention claim. Proposed compounds can be used in medicinal practice in treatment and prophylaxis of cardiovascular diseases.

EFFECT: valuable medicinal properties of compounds.

7 tbl, 1 dwg, 3 ex

Description

The invention relates to new chemical compounds of the heterocyclic series, namely, derivatives of 2-H-1-benzopyran-2-one of the general formula I

where R = —CH ₃ , R ₁ = —NH— (CH ₂ ) ₂ —Br (1); R = -CH ₃ , R ₁ = -NH- (CH ₂ ) ₃ -Cl (2);

R = -CH ₃ , R ₁ = -O- (CH ₂ ) ₂ -Br (3); R = -CH ₃ , R ₁ = -O- (CH ₂ ) ₃ -Cl (4);

R = -H, R ₁ = -O- (CH ₂ ) ₂ -Br (5); R = -H, R ₁ = -O- (CH ₂ ) ₃ -Cl (6);

R = -O- (CH ₂ ) ₂ -Br, R ₁ = -H (7); R = -O- (CH ₂ ) ₃ -Cl, R ₁ = -H (8);

R = -H, R ₁ = -O- (CH ₂ ) ₂ -OH (9); R = -H, R ₁ = -O- (CH ₂ ) ₂ -SH (10);

R = -H, R ₁ = -O- (CH ₂ ) ₂ -NH ₂ (11); R = -CH ₃ , R ₁ = -O- (CH ₂ ) ₂ -NH ₂ (12);

R = -CH ₃ , R ₁ = -O- (CH ₂ ) ₂ -O- (CH ₂ ) ₂ -OH (13);

R = -H, R ₁ = -O- (CH ₂ ) ₂ -O- (CH ₂ ) ₂ -OH (14);

which have varying degrees of anticalcium activity and may find application in medical practice in the treatment of cardiovascular diseases.

In medical practice, in the treatment of cardiovascular insufficiency, a number of drugs are often used: finoptin, various dosage forms of nifedipine, diltiazem, amlodipine, etc., with short and prolonged anticalcium activity (Mashkovsky MD Medicines. - 2000. - T.1 - S.413-415; Kuleshova E.V. Calcium antagonists and their role in the treatment of diseases of the cardiovascular system. - 2002. - Part 1 and 2).

However, the above drugs have a high frequency of side effects, in particular finoptin up to 10%, and nifedipine up to 30%, manifested in the form of swelling of the legs, hypotension, changes in heart rate, etc. In addition, in recent years it has been noted that the use of short-acting dihydropyridines, especially nifedipine, may increase the risk of developing malignant tumors, myocardial infarction and death in patients with arterial hypertension (Kuleshova E.V. Calcium antagonists and their role in the treatment of heart disease udistic system. - 2002. - Part 1 and 2; Furberg CD, Psaty B.M., Meyer JV Circulation. - 1995. - V.92. - P.1326-1331; Psaty V.M., Heckbert SR, Koepsell T .D. Et al. JAMA. - 1995. - V.274. - P.620-625; Pahor M., Psaty BM, Alderman MH et al. Lancet. - 2000. - Dec. 9. - 356 (9246) : 1970-4). In animal experiments, embryotoxicity of diltiazem and most 1,4-dihydropyridine series antagonists was also established. With prolonged therapy with diltiazem, nifedipine and felodipine in early pregnancy, cases of fetal death and abnormalities of the skeleton in newborns are described. Therefore, diltiazem and all 1,4-dihydropyridine derivatives of the first and second generation are contraindicated in any period of pregnancy (Svensson A. Clin. Exp. Hypertension. - 1993. - V.15. - P.1353-1361; Lowe SA, Rubin P. S. J. Hypertension. - 1992. - V.10. - P.201-207).

In connection with the foregoing, the synthesis and study of new antagonists of calcium ions from other groups of heterocyclic compounds (not 1,4-dihydropyridine series) is very relevant for practical medicine, especially for clinical cardiology.

The closest in structure, physico-chemical and pharmacological properties to the claimed group of compounds is 7,7'-ethylenedioxy-2-H-1-benzopyran-2,2'-dione-diumancal (15), previously synthesized and patented by us in Russia as an anti-ischemic agent (Abyshev A.Z., Denisenko P.P., Pukhov M.P. Patent of Russia No. 1836086; Abyshev A.Z., Dyachuk G.I. Patent of Russia No. 2155036; Abyshev A.Z., Agaev E .M., Marishchenko O.V. Patent of Russia No. 2187303).

diumancal-7,7'-ethylenedioxy-2-H-1-benzopyran-2,2'-dione (15)

This compound is approved by the Pharmacological State Committee of the Ministry of Health of the Russian Federation (protocol No. 10 dated 06/27/96), and by order of the Minister of Health it is approved for use in medical practice in the form of tablets of 0.01 g [Ankardin (diumancal) - forte] for the treatment of coronary heart disease in adults (order No. 202 of 07/14/97).

Thus, in the course of many years of research among oxygen-containing heterocyclic compounds, we first discovered a substance - diumancal (15), which has anticalcium activity and differs from calcium antagonists of phenylalkylamine and 1,4-dihydropyridine series in higher activity (30 mg / day), therapeutic latitude (25000) and low toxicity (LD ₅₀ = 2500 mg / kg).

It should be noted that the representative of phenylalkylamines - finoptin is active in doses of 240-480 mg / day., 1,4-dihydropyridines - nifedipine - 30-60 mg / day. and benzothiazepines - diltiazem - 240-360 mg / day.

The aim of the present invention is to search for new compounds of the 2-H-1-benzopyran-2-one series - diumancal analogues having more pronounced anticalcium activity and safety than compounds used in medical practice, namely phenylalkylamines, 1,4-dihydropyridines and benzothiazepines .

This goal is achieved by the synthesis and study of the anticalcium properties of previously unknown compounds of the series 2-H-1-benzopyran-2-one - analogues of diumancal (15),

To date, a large number of similar compounds have been synthesized, and for many of them the physicochemical and pharmacological properties have been studied. Therefore, the invention is illustrated by the following specific examples of the synthesis and study of the anticalcium activity of some of them.

Example 1. General procedure for the synthesis of amino, thio - or oxyalkyl derivatives of 2-H-1-benzopyran-2-one

A. In a three-necked round-bottomed flask with a capacity of 300 ml, equipped with a mechanical stirrer, a reflux condenser with a calcium chloride tube and a dropping funnel, 150 ml of ethylene glycol (EG) or dimethylformamide (DMF), corresponding weights of 4-hydroxy, 7-hydroxy, 4- methyl 7-hydroxy, 4-methyl-7-amino or 4,6-dimethyl-7-amino-benzopyran-2-one and potassium carbonate. The reaction mixture is heated in an oil bath (from 80 ° C to 110 ° C) with constant stirring for 2 hours. After dissolving weighed portions of the compounds, appropriate amounts of 1,2-dibromoethane, 1-bromo-3-chloropropane *, 1-chloro-2-aminoethane or 1-chloro-2-mercaptoethane are added dropwise and the mixture is continued to heat with stirring for another 16 hours, after which is cooled to room temperature and water is added. The resulting crystals are filtered off, washed thoroughly with water and the residue is recrystallized from various solvents. In this case, individual compounds are obtained — 15, 16, 18, 21, 22, 24, 28, 30, the physicochemical parameters of which are presented in Table 1.

* Bromo or chloralkyl derivatives containing 4 to 6 carbon atoms in the side chain were also synthesized.

B. Stock solutions are concentrated in vacuo and applied to a column (3 × 80 cm) filled with L 100/250 silica gel. Elution is carried out with petroleum ether (mp. 40-70 ° C), a mixture of the latter with chloroform and chloroform, collecting fractions of 100 ml. After concentration of the fractions and recrystallization of the precipitated residues from the corresponding solvents, compounds 1-14 are obtained in an individual form. Their physico-chemical characteristics are also presented in table 1.

Example 2. General procedure for the synthesis of combined compounds 17, 19, 20, 23, 25-27.29, 31-36.

C. In a two-neck round-bottomed flask with a capacity of 250 ml, equipped with a mechanical stirrer, reflux condenser with a calcium chloride tube, 100 ml of DMF, corresponding weights of 4-hydroxy, 7-hydroxy, 4-methyl-7-aminoethyl-, 4,6- are placed dimethyl-7-amino or 6-glucosyl-7-hydroxy-benzopyran-2-one and potassium carbonate. The reaction mixture is heated in an oil bath (from 80 ° C to 110 ° C) with constant stirring for 2 hours. After dissolution of the weighed portions of the compounds, the corresponding weighed amounts of 7- (2'-bromethyloxy) -, 4-methyl-7- (2'-bromethyloxy) - or 4-methyl-7- (2'-bromoethylamino) benzopyran-2-one are added and the mixture is continued to heat with stirring for another 5 hours, after which it is cooled to room temperature and water is added. The resulting crystals are filtered off, washed thoroughly with water and the residue is recrystallized from various solvents. In this case, individual compounds are obtained - 17, 19, 20, 23, 25-27, the physicochemical parameters of which are presented in Table 1.

D. Compounds 28-36 are also synthesized under conditions B by reacting the corresponding 7- (2'-hydroxyethyloxy) -, 7- (2'-sulfohydroethyloxy) - or 7- (2'-aminoethyloxy) derivatives of benzopyran-2-one or 4-methyl-benzopyran-2-one with 7- (2'-bromethyloxy) - or 4-methyl-7- (2 'bromethyloxy) benzopyran-2-one. Their physico-chemical characteristics are presented in table 1.

Example 3. Determination of anticalcium activity of the synthesized compounds.

Due to the fact that many calcium antagonists (AK) are most often used as antiarrhythmic drugs, a number of arrhythmias of various genesis were used in our work. In particular, the primary screening of the studied groups of compounds was carried out in mice using a model of calcium chloride arrhythmia. With the introduction of excess calcium chloride, arrhythmia manifests itself in the form of fibrillation, ventricular flutter, followed by cardiac arrest. In this experiment, a dose of calcium chloride of 330 mg / kg was used, which was administered intravenously (iv), causing 100% death of mice during the first 20-40 seconds. The studied compounds were administered intraperitoneally 2, 5, and 15 min before the iv administration of calcium chloride. Verapamil, diltiazem, and nifedipine were used as comparison drugs, and the ED ₅₀ value was recorded according to the mouse survival test.

The data presented in Table 2 show that the ED ₅₀ for verapamil is 10 mg / kg, diltiazem is less than 0.1 mg / kg, and nifedipine in the studied doses provided little protection against excess calcium ions. The ED ₅₀ for some test compounds was, for example, for AK-5 less than 0.01 mg / kg, AK-3 and AK-15 0.1 mg / kg, AK-13 1.0 mg / kg, AK-17 10 , 0 mg / kg, etc. The LD _{50 of the} studied compounds was also determined, and it is noteworthy that most of the compounds under consideration were found to be slightly toxic.

A comparison of the indicators given in Table 2 (ED ₅₀ / LD ₅₀ ) shows that the breadth of the therapeutic effect of many synthesized diumancal analogues (15) is significant and exceeds that of the known calcium antagonists. In addition, it can also be seen from the data presented in Table 2 that, on the calcium chloride model of arrhythmia, the protective index of a number of substances (AK-6, 20, 22, 25, etc.) is superior to that of the known AK - verapamil and diltiazem. In this case, the greatest activity activity was observed for compound AK-22.

Analysis of the data obtained shows that, regardless of the origin and method of synthesis of 4- or 7-monosubstituted, 4,7-disubstituted or dimeric derivatives of 2-H-1-benzopyran-2-one, interconnected in positions -7.7 ' various alkyl or aminoalkyl radicals have been found to be practically non-toxic. 5 and 7-alkyl- or alkylhalo derivatives of 2-H-1-benzopyran-2-one and their analogues have an LD _{50 of} about 800 mg / kg, and 4-hydroxy or 4-methyl-2-H-1-benzopyran-2 - they are 1000 mg / kg.

Further study of the most effective compounds was carried out on other models of experimental arrhythmias caused by various pharmacological drugs, but in comparison, mainly with verapamil.

Thus, it was shown that pituitrin (1 mg / kg) administered to intact mice causes arrhythmias and conduction disturbance in 100% of cases (see Table 3). Verapamil (10 mg / kg), administered 30 minutes before the iv injection of pituitrin, did not eliminate arrhythmia, but stopped conduction disturbances in 50% of animals, while among the synthesized new compounds AK-26 and 34 (1 mg / kg) like verapamil, conduction disturbances were prevented in 60% and 30% of mice, and, in addition, in 35% and 20% of cases, the development of arrhythmia was arrested.

In the genesis of strophanthin arrhythmia are processes of violation of the permeability of cell membranes for K ⁺ and Ca ^{2+ ions} . With the introduction of strophanthin at a dose of 0.06 mg / kg in 100% of mice, arrhythmia is observed, in 80% of conduction disturbances and death of 30% of animals is noted (see Table 4). Verapamil (10 mg / kg), administered 30 minutes before the iv injection of strophanthin, prevented the death of animals, the appearance of arrhythmias and impaired conduction. Almost all the studied compounds prevented the death of animals, and had an effect similar to verapamil, but in a lower dose.

From the data presented in Tables 2-4, it follows that antiarrhythmic activity is mainly expressed in 2-H-1-benzopyran-2-ones substituted only at position 7 (diumankal and its analogues), the most active of which are substances containing between two benzopyran rings no more than two oxymethylene groups.

Based on the above data, it can be concluded that the biological activity of the synthesized derivatives of 2-H-1-benzopyran-2-one substantially depends on the structure and location of the side chains (alkyl, hydroxyalkyl, haloalkyl).

An important role is also played by the presence in the structure of 2-H-1-benzopyran-2-one of the conjugated system —CH = CH— in the α-pyran ring, but the latter, apparently, is not a decisive factor, since unsubstituted 2-H -1-benzopyran-2-one and its 3-carboxy- and 4-hydroxy derivatives containing this system in their molecules are less active than other studied compounds of this series.

Thus, the results obtained show that the studied derivatives of 2-H-1-benzopyran-2-one, i.e. Diumancal analogues, by their mechanism of action, can be assigned to the group of new generations of calcium ion antagonists, which was further confirmed by us using various methodological techniques.

1. The data presented in Table 5 show that the most significant inhibition of the incorporation of ⁴⁵ Ca in the nerve cells of rat brain is observed with the action of diumancal (AK-15), AK-27 and AK-28, while with the introduction of verapamil a significant effect is noted in a dose that differs by an order of magnitude.

2. The selective action of 2-H-1-benzopyran-2-one derivatives, in particular diumancal (AK-15) and its analogues, on the pool of calcium ions in nerve terminals was established by us using the pyroantimonate method for detecting calcium granules. The results showed that the excess of calcium ions that occurs in the nerve terminals under the action of the convulsive agent norbornane is eliminated by the administration of diumancal (see drawing).

Thus, it was found for the first time that the studied group of compounds, as antagonists of calcium ions, actively influence not only the conductivity potential of dependent L-type calcium channels, but also exhibit pronounced sensitivity to N- and T-type channels, widely represented in the central nervous system and in the ganglia.

3. The ability of some studied derivatives of 2-H-1-benopyran-2-one (AK-15, 18, 20, 31, 33, 36, etc.) to inhibit the flow of calcium ions through membranes was also established when recording the kinetics of calcium channels on isolated clam neurons.

During the implementation of this section of the work, the object of study was the neurons of the gastropod mollusk - the large pond (Lymnaea stagnalis), since the ionic membrane mechanisms of electrogenesis and the functioning patterns of the transmembrane ion channels of mollusk neurons are fundamentally similar to those of the neurons of other animals, including mammals. The possibility of isolating isolated nerve cells, their large sizes, and long-term functioning in experiments (up to 1.5 hours) allow us to study in detail the mechanisms of generating electrical potentials, as well as electrical phenomena at the level of potential of dependent ion channels of an excitable membrane under the action of pharmacological agents.

To measure transmembrane ion currents, the method of intracellular perfusion of isolated neurons and fixation of the membrane potential (MP) was used. This method allows for the complete replacement of the intracellular medium in isolated cells by dialysis through a destroyed portion of the surface membrane, to control and arbitrarily change all concentration gradients on the membrane of an isolated cell, to act with pharmacological preparations in different concentrations on both sides of the membrane, and also to measure transmembrane ion currents under conditions fixing potential without the use of microelectrodes.

For differentiation of ion currents, media with different ionic composition were used, and the phenomenon of different potential dependence of ionic currents was used. All test substances were added to external (perfusion) solutions in increasing concentrations from 0.0001 to 10,000 μM / L.

The separation of pure ion currents with their stable parameters, which were taken as the initial values (before the action of the substance), occurred 3-5 minutes after the complete replacement of the corresponding solutions. At the same time, an isolated current with a characteristic inactivation phase appeared on the oscilloscope screen.

In our studies, it was found that all the studied compounds with extracellular application reduced the amplitude of both the incoming and outgoing ion currents. This effect was dose dependent. As the concentration of the studied substances increased, an ever greater suppression of transmembrane isolated ionic currents was observed.

The results are presented in tables 6 and 7.

To confirm the relative selective effect of the studied compounds on the incoming calcium currents, we obtained calculated values of the effective concentrations of 50% suppression (EC ₅₀ ) of transmembrane ion currents.

Comparison of EC ₅₀ values showed that for 50% suppression of calcium current the lowest concentrations of the studied substances are necessary. Based on these data, we obtained series of activity of the studied compounds with respect to each ion current.

According to the degree of suppression of calcium currents, these compounds can be arranged in the following order:

AK-20> DIUMANKAL> VERAPAMIL> AK-36> AK-31> AK-33> AK-18.

The same arrangement is observed with respect to the sodium current.

Literature data (Krutetskaya Z.I., Lonsky A.V. Membrane biophysics. - St. Petersburg. - 1994. - 206 p .; S. Kosterin. Calcium transport in smooth muscles. - Kiev. - “Naukova Dumka”. - 1990. - 216 pp.) Indicate that the potential dependent channels of various types have a similar molecular structure. This, in our opinion, explains the similar dynamics in the action of the studied substances, however, the calculation of the effective concentrations of 50% suppression demonstrates that 50% suppression of the sodium current is observed at concentrations exceeding those for calcium by 8.5 or more times. These data allow us to conclude that the studied compounds exhibit more selective activity with respect to the incoming calcium current compared to other currents (incoming sodium and outgoing potassium). This is not valid for compounds AK-18 and AK-31, in which the difference between the EU ₅₀ between calcium and sodium currents is 1.02 and 1.59, respectively.

Thus, the most probable ways of realizing the action of the studied compounds are direct blockade at the entrance gates of slow calcium channels, competition with Ca ²⁺ for the active centers of these gates, deformation of the entire structure of the calcium transport channel that impedes the normal passage of Ca ²⁺ through them, non-specific properties to stabilize the cell membrane.

The possibility of the interaction of the studied substances with the gate mechanisms of the channels cannot also be rejected, since a decrease in ion currents is also possible due to a decrease in the open time of individual channels or a decrease in the frequency of their opening. This may be evidenced by the fact that the kinetics of the calcium transmembrane ion current changes under the influence of the AK-20 compound. Since the rate of activation or inactivation of ion currents is determined, first of all, by the degree of functional activity of the corresponding gate structures (activation m-gates or inactivation h-gates) of ion channels, it is possible that this compound can interact directly with the gate particles of calcium channels. Under the influence of the remaining compounds studied, the kinetics of the studied transmembrane ion currents remained virtually unchanged over the entire concentration range (see Tables 6 and 7).

Despite such a complex and ambiguous nature of the relationship between transmembrane ion currents, we can nevertheless conclude that the leading mechanism of action of the compounds studied is the blockade of Ca ²⁺ entry into the cell through slow calcium channels, i.e. the studied compounds really belong to the group of new generation calcium ion antagonists.

Thus, based on the presented experimental data, we confirmed the discovery of a completely independent class of new generation calcium ion antagonists from a number of 2-H-1-benzopyran-2-one derivatives that actively influence the conductivity of the potential of calcium-dependent channels not only of L-type, widely represented in the muscles of the heart and blood vessels, but also of the N- and T-types, the most widely distributed in the central nervous system and ganglia. It should be emphasized that the known antagonists of calcium ions of the phenylalkyl, 1,4-dihydropyridine and benzothiazepine series do not possess such a wide spectrum of action.

Claims (1)

Derivatives of 2-H-1-benzopyran-2-one of general formula I

where R = —CH ₃ , R ₁ = —NH— (CH ₂ ) ₂ —Br (1); R = -CH ₃ , R ₁ = -NH- (CH ₂ ) ₃ -Cl (2); R = -O- (CH ₂ ) ₂ -Br, R ₁ = -H (7); R = -O- (CH ₂ ) ₃ -Cl, R ₁ = -H (8); R = -H, R ₁ = -O- (CH ₂ ) ₂ -SH (10); R = -H, R ₁ = -O- (CH ₂ ) ₂ -NH ₂ (11); R = -CH ₃ , R ₁ = -O- (CH ₂ ) ₂ -NH ₂ (12); R = -H, R ₁ = -O- (CH ₂ ) ₂ -O- (CH ₂ ) ₂ -OH (14);

showing anti-calcium activity.